DNA aptamers for promoting remyelination

ABSTRACT

Materials and methods related to using multimeric DNA aptamers to treat demyelinating diseases are provided herein.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a National Stage application under 35 U.S.C. 371 ofInternational Application No. PCT/US2012/035005, having an InternationalFiling Date of Apr. 25, 2012, which claims benefit of priority from U.S.Provisional Application Ser. Nos. 61/565,144, filed on Nov. 30, 2011,and 61/480,179, filed Apr. 28, 2011, each of which is incorporatedherein by reference in its entirety.

TECHNICAL FIELD

This document relates to materials and methods for reducingdemyelination and promoting remyelination, and to the use of multimericDNA aptamers to treat demyelinating disease.

BACKGROUND

Multiple sclerosis (MS) is a debilitating inflammatory disease of thecentral nervous system (CNS) that is characterized by local destructionof the insulating myelin surrounding neuronal axons (Compston and Coles(2002) Lancet 359:1221-1231). With more than 200 million MS patientsworldwide, there is great need for effective treatments that preventprogression or induce repair. Anti-inflammatory therapies have met withsome success in preventing relapses (Bates (2011) Neurol. 76:S14-25).Some naturally occurring IgM antibodies identified from human serum canpromote both cell signaling and remyelination of CNS lesions in an MSmodel involving chronic infection of susceptible mice by Theiler'sencephalomyelitis virus (Warrington et al. (2000) Proc. Natl. Acad. Sci.USA 97:6820-6825) and in the lysolecithin model of focal demyelination(Bieber et al. (2002) Glia 3:241-249). This result raised thepossibility that molecules with binding specificity for oligodendrocytesor myelin components may promote therapeutic remyelination in MS.

SUMMARY

This document is based in part on the identification of a 40-nucleotide,single-stranded DNA aptamer that has affinity for murine myelin and canpromote remyelination in a model of MS. As described below, the aptamercan bind to myelin in vitro and in live cerebellar cultures. Peritonealinjection of a formulation containing the aptamer promoted remyelinationof CNS lesions in mice infected by Theiler's virus. Interestingly, theDNA aptamer contains guanosine-rich sequences predicted to induceintramolecular folding or intermolecular assembly involving guanosinequartet structures. Relative to monoclonal antibodies, DNA aptamers aresmall, stable, and non-immunogenic, suggesting new possibilities for MStreatment.

In one aspect, this document features a method for promoting neuronalremyelination in a subject in need thereof. The method can includeadministering to the subject a pharmaceutical composition comprising amultimeric nucleic acid aptamer in an amount effective to promoteremyelination. The nucleic acid aptamer can be a homotetramer. Thenucleic acid aptamer can have the sequence set forth in SEQ ID NO:17.The remyelination can be mediated by central nervous system-type myelinproducing cells (oligodendrocytes) or mediated by peripheral nervoussystem-type myelin producing cells (Schwann cells). The subject can bediagnosed with a demyelinating disease (e.g., a demyelinating diseaseselected from the group consisting of multiple sclerosis, idiopathicinflammatory demyelinating diseases, transverse myelitis, Devic'sdisease progressive multifocal leukoencephalopathy, optic neuritis,leukodystrophies and acute disseminated encephalomyelitis,Guillain-Barré syndrome, chronic inflammatory demyelinatingpolyneuropathy, anti-MAG peripheral neuropathy, and Charcot-Marie-Toothdisease).

In another aspect, this document features a composition containing apharmaceutically acceptable carrier and a nucleic acid aptamercontaining the sequence set forth in SEQ ID NO:17. The nucleic acidaptamer can be a homotetramer.

In another aspect, this document features the use of a multimericnucleic acid aptamer in the treatment of a demyelinating disease. Themultimeric nucleic acid aptamer can contain the sequence set forth inSEQ ID NO:17. The multimeric nucleic acid aptamer can be a homotetramer.The demyelinating disease can be selected from the group consisting ofmultiple sclerosis, idiopathic inflammatory demyelinating diseases,transverse myelitis, Devic's disease progressive multifocalleukoencephalopathy, optic neuritis, leukodystrophies and acutedisseminated encephalomyelitis, Guillain-Barré syndrome, chronicinflammatory demyelinating polyneuropathy, anti-MAG peripheralneuropathy, and Charcot-Marie-Tooth disease.

This document also features the use of a multimeric nucleic acid aptamerin the preparation of a medicament for treating a demyelinating disease.The multimeric nucleic acid aptamer can contain the sequence set forthin SEQ ID NO:17. The multimeric nucleic acid aptamer can be ahomotetramer. The demyelinating disease can be selected from the groupconsisting of multiple sclerosis, idiopathic inflammatory demyelinatingdiseases, transverse myelitis, Devic's disease progressive multifocalleukoencephalopathy, optic neuritis, leukodystrophies and acutedisseminated encephalomyelitis, Guillain-Barré syndrome, chronicinflammatory demyelinating polyneuropathy, anti-MAG peripheralneuropathy, and Charcot-Marie-Tooth disease.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. Although methods and materialssimilar or equivalent to those described herein can be used to practicethe invention, suitable methods and materials are described below. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willcontrol. In addition, the materials, methods, and examples areillustrative only and not intended to be limiting.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1 is a schematic showing steps in the selection of DNA aptamersspecific for components of crude mouse myelin suspension. FIG. 1A: Apool of about 10¹⁵ 100-nucleotide fluorescent (*) single-stranded DNAaptamers containing 60 nucleotides of random sequence was generated by aPCR and mixed with a suspension of crude mouse myelin. Cycles ofaffinity purification and amplification yielded a myelin-specific DNAaptamer. FIG. 1B: Deoxyguanosine-rich DNA aptamer 3064 (SEQ ID NO:17)specific for crude mouse myelin is shown folded as a putativeintramolecular G-quadruplex. FIG. 1C: Tetravalent complex of3′-biotinylated DNA aptamer (thin lines) with streptavidin (circles).FIG. 1D: Negative control deoxyguanosine-rich DNA aptamer 3060 (SEQ IDNO:20) specific for chelated nickel ions, shown as putativeintramolecular G-quadruplex. FIG. 1E: Negative controloligodeoxythymidylate aptamer 3202 (SEQ ID NO:21).

FIG. 2 is a diagram showing chemical modifications of DNA aptamersrelevant to the work described herein. FIG. 2A shows the 5′-fluoresceinmodification that was used for in vitro selection and binding studies.FIG. 2B shows the 3′ biotin modification that was present in aptamersused for in vivo injection.

FIG. 3 is a graph plotting the results of in vitro selection of DNAaptamer pools over 10-11 rounds of selection and amplification. Targetswere myelin oligodendrocyte glycoprotein (MOG) immobilized on Ni-NTAmagnetic beads (filled circles) or suspension of crude mouse myelin inbuffer (open circles). MOG selections gave rise to aptamer 3060(selective for chelated Nickel beads). Myelin selections gave rise toaptamer 3064.

FIG. 4 shows the nucleotide sequences of anti-myelin DNA aptamers andderived sub-sequences. The top panel shows initial sequences of DNAaptamers after cloning. Sequences derived from random regions are inregular font. Fixed sequences for PCR primer binding are underlined. Thetop three sequences correspond to aptamer 3028. The bottom panel showsthe sequences of aptamer sub-sequences derived from anti-myelin aptamer3028.

FIG. 5A is a graph plotting binding of fluorescent aptamers 3064 (filledcircles), 3060 (filled squares), and 3202 (filled triangles) to crudemouse myelin proteins as detected by sedimentation of the insolublemyelin fraction. Mean and standard deviation are shown for threeexperiments. FIG. 5B is an image showing lanes from gels and blotsdemonstrating DNA aptamer specificity. Lane 1: Coomassie staining ofcrude myelin proteins (myelin; CBB) separated by SDS-polyacrylamide gelelectrophoresis. Lanes 2-7: results of western and southwestern blottingwith the indicated antibodies or fluorescent aptamers. Mobilities ofmolecular weight standards are indicated at left, and apparent molecularweights of aptamer-reactive proteins are indicated at right.

FIG. 6 is a series of images showing aptamer staining of unfixedcerebellar slices. Live mouse cerebellar slices were incubated with 1 μMfluorescein labeled aptamer in PBS for 30 minutes on ice. The tissue wasthen washed, fixed with 4% paraformaldehyde and mounted. Binding of thefluorescein-labeled aptamers to myelin rich regions of the cerebellumwas detected as fluorescein epi-fluorescence images with an OlympusDP-70 camera (FL, upper panels). Matching phase contrast images areshown in the lower panels (PH). Background fluorescence showed nospecific signal. Aptamer 3060 (nickel specificity) showed diffusebinding throughout the molecular cell layer of the cerebellum. Aptamer3064 demonstrated myelin specific binding to white matter tracts of thecerebellum. Control aptamer 3202 (poly-dT) showed less binding.

FIG. 7 is a schematic illustrating the experimental approach for in vivostudies. After cloning and identification of the DNA subsequenceimportant for myelin binding, DNA aptamers were injected into theperitoneal cavities of mice with demyelinating CNS lesions induced byTheiler's encephalomyelitis virus infection. Immunopathology at 7-9months after infection revealed enhanced remyelination.

FIG. 8 is an image showing formation of 3′-biotinylated DNA aptamermultimers by incubation with streptavidin. The indicated5′-fluoresceinated, 3′-biotinylated DNA aptamers (lanes 4, 8, and 12)were folded and then incubated with different amounts of streptavidin toproduce multimeric aptamer complexes. Aptamer:streptavidin concentrationratios were 1:1 (lanes 1, 5, and 9), 4:1 (lanes 2, 6, and 10), and 20:1(lanes 3, 7, and 11). Mobilities of aptamer monomer and complexescontaining one to four aptamers (1-4) are indicated. Complexes with twobound aptamers displayed two distinct mobilities due to cis vs. transbinding arrangements on the streptavidin tetramer.

FIG. 9A is a series of three representative neuropathology micrographsfrom spinal cords of mice infected with Theiler's virus and treated withvarious aptamers. Left panel: normal appearing white matter (WM).Center: demyelination (DM). Right: remyelination (RM). After blindedmicrograph review of specimens from control and aptamer-treated animals,the percent of spinal cord quadrants showing demyelination orremyelination was determined. Results are reported in Table 1 herein.FIG. 9B is a series of light photomicrographs demonstrating examples ofTMEV-mediated spinal cord demyelination (lower panels) and remyelination(upper panels) in mice treated with the indicated DNA aptamers. Thescale bar in lower right panel is 100 micrometers. After blindedmicrograph review of specimens from control and aptamer-treated animals,the percent of spinal cord quadrants showing demyelination orremyelination was determined as reported in Table 2.

DETAILED DESCRIPTION

MS is a debilitating neurological disease with a prevalence of about0.1% in the Western world (Mayr et al. (2003) Neurol. 61:1373-1377). MSis fundamentally an inflammatory disease that leads to CNS lesionscharacterized by the loss of myelin required for electrical insulationof neuronal axons (Noseworthy et al. (2000) New Engl. J. Med. (2000)343:938-952). The resulting symptoms, including fatigue, gaitimpairment, cognitive impairment, and vision loss, can lead to permanentdisability (Rodriguez (1994) Neurol. 44:28-33).

While the origin of MS remains unresolved, therapy and cure present evengreater challenges. Therapies for relapsing MS include plasma exchangeto remove pathogenic immunoglobulins and/or treatment withanti-inflammatory drugs such as glatiramer acetate, β interferon,mitoxantrone, and natalizumab (Bates, supra). These approaches are notcurative, and are ineffective in some cases (Freedman (2011) Neurol.76:S26-34). It remains unclear whether curative therapy should bedirected against the immune system, or toward repair and rescue ofoligodendrocytes and myelin.

Previous studies identified multiple natural murine and human IgMautoantibodies that can bind to live cerebellum and culturedoligodendrocytes and promote remyelination in mice (Warrington et al.,supra). Target antigens are not known in molecular detail, but thepentavalent character of the IgM antibody is important for activity (PazSoldan et al. (2003) Mol. Cell. Neurosci. 22:14-24).

This document provides myelin-binding agents that are smaller and morerobust than the previously identified IgM monoclonal antibodies. Inparticular, as described in the Examples below, an in vitro selectionmethod was used to identify small, single-stranded DNA aptamers thathave affinity for myelin and that can promote remyelination in mice.Aptamers are folded, single-stranded nucleic acids with activities that,like folded proteins, depend on their three-dimensional shapes andsurface features. The advantages of aptamers can, in some embodiments,include one or more of the following: small size, chemical stability,ease of synthesis, lack of immunogenicity, and the availability of invitro selection technology in which cycles of affinity selection andamplification can be used to identify nucleic acids with rare propertiesfrom random libraries that can contain 10¹⁴ or more candidates—chemicaldiversity exceeding that encoded in mammalian immune systems.

Aptamers useful for promoting remyelination can be about 10 to about 50nucleotides in length (e.g., 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or 51 nucleotides inlength, or any range there between), and can include, for example thosehaving the sequences shown in FIG. 4 (SEQ ID NOS:3 to 19) or fragmentsthereof. An aptamer containing, consisting of, or consisting essentiallyof the sequence 5′-ATACCAGCTTATTCAATTGGGTCGGCGGGTGGGGTGGGAGGTGGTCTTGTCTCTGGGTTTTGTTGTGAACCACACGTAAGATAGTAAGTGCAATCT-3′ (SEQ ID NO:5) or5′-GGGTCGGCGGGTGGGGTGGGAGGTGGTCTTGTCTCTGGGT-3′ (SEQ ID NO:17), or afragment thereof, can be particularly useful.

A nucleic acid aptamer can be monomeric, or can be multimeric. Forexample, an aptamer can be a dimer, a trimer, a tetramer, a pentamer, ora hexamer, and can be homomeric or heteromeric. In some embodiments, anaptamer can be configured as a homomultimer. See, for example, theExamples below, which describe the use of a homotetramer containing fourcopies of SEQ ID NO:17. In some cases, an aptamer can be complexed withanother compound that can provide stabilization and/or multimerization.For example, one or more aptamers can be combined using biotin-avidinlinkages as described in the Examples, or with polyethylene glycollinkages (see, e.g., Govan et al. (2011) Bioconjugate Chem.22(10):2136-2142). In some embodiments, aptamer synthesis can beperformed with a solid support to yield structures in which the aptamersare head-to-head multimers. Synthesis reagents are availablecommercially (e.g., from Glen Research; Sterling, Va.); see, alsoShchepinov (1999) The Glen Report 12(1):1-4, available on the World WideWeb at glenresearch.com/GlenReports/GR12-11.html andglenresearch.com/GlenReports/GR12-1.pdf. Further, azide/alkyne 3+2cycloaddition chemistry can allow for rapid coupling of derivatizedoligonucleotides to a central chemical backbone. Such a procedure couldbe used to create oligonucleotide multimers cleanly and efficientlywithout a central protein. See, e.g., Rostovtsev et al. (2002) AngewChem. Int. Ed. 41:2596-2599; and Huisgen (1963) Angew Chem. Int. Ed.2:565-598, as well as The Glen Report (2008) 20(1):8-9 available on theWorld Wide Web at glenresearch.com/GlenReports/GR20-14.html andglenresearch.com/GlenReports/GR20-1.pdf. See, also, U.S. Publication No.2009/0234105; Alleti et al. (2010) J. Org. Chem. 75(17):5895-5903; andYim et al. (2010) J. Med. Chem. 53(10):3944-3953.

The aptamers described herein can be used to treat demyelinatingdiseases (e.g., MS) by inducing remyelination. For example, a nucleicacid aptamer as described herein (e.g., an aptamer having the sequenceset forth in SEQ ID NO:17) can be synthesized and formulated into apharmaceutical composition for administration to a subject diagnosed ashaving a disorder of the nervous system in which the myelin sheath ofneurons is damaged. In addition to MS, demyelinating diseases that canaffect the central nervous system include idiopathic inflammatorydemyelinating diseases, transverse myelitis, Devic's disease progressivemultifocal leukoencephalopathy, optic neuritis, and leukodystrophies andacute disseminated encephalomyelitis (ADEM). Demyelinating diseases thatcan affect the peripheral nervous system include Guillain-Barrésyndrome, chronic inflammatory demyelinating polyneuropathy, anti-MAGperipheral neuropathy, and Charcot-Marie-Tooth disease.

The aptamers described herein can be incorporated into compositions foradministration to a subject in need thereof (e.g., a subject identifiedas having a demyelinating disease). Thus, this document provides, forexample, the use of aptamers as described herein in the manufacture ofmedicaments for treating (e.g., reducing) demyelination and/or enhancingremyelination.

Methods for formulating and subsequently administering therapeuticcompositions are well known to those in the art. Dosages typically aredependent on the responsiveness of the subject to the compound, with thecourse of treatment lasting from several days to several months, oruntil a suitable response is achieved. Persons of ordinary skill in theart routinely determine optimum dosages, dosing methodologies andrepetition rates. Optimum dosages can vary depending on the relativepotency of an aptamer, and generally can be estimated based on the EC₅₀found to be effective in in vitro and/or in vivo animal models.Compositions containing the aptamers may be given once or more daily,weekly, monthly, or even less often, or can be administered continuouslyfor a period of time (e.g., hours, days, or weeks). For example, anaptamer or a composition containing an aptamer can be administered to apatient at a dose of at least about 0.01 ng/kg to about 100 mg/kg ofbody mass.

An aptamer can be admixed, encapsulated, conjugated or otherwiseassociated with other molecules, molecular structures, or mixtures ofcompounds such as, for example, liposomes, receptor or cell targetedmolecules, or oral, topical or other formulations for assisting inuptake, distribution and/or absorption.

In some embodiments, a composition can contain an aptamer as providedherein in combination with a pharmaceutically acceptable carrier.Pharmaceutically acceptable carriers include, for example,pharmaceutically acceptable solvents, suspending agents, or any otherpharmacologically inert vehicles for delivering nucleic acid aptamers toa subject. Pharmaceutically acceptable carriers can be liquid or solid,and can be selected with the planned manner of administration in mind soas to provide for the desired bulk, consistency, and other pertinenttransport and chemical properties, when combined with one or moretherapeutic compounds and any other components of a given pharmaceuticalcomposition. Typical pharmaceutically acceptable carriers include,without limitation: water; saline solution; binding agents (e.g.,polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e.g.,lactose or dextrose and other sugars, gelatin, or calcium sulfate);lubricants (e.g., starch, polyethylene glycol, or sodium acetate);disintegrates (e.g., starch or sodium starch glycolate); and wettingagents (e.g., sodium lauryl sulfate).

Pharmaceutical compositions containing aptamers as described herein canbe administered by a number of methods, depending upon whether local orsystemic treatment is desired. Administration can be, for example,parenteral (e.g., by subcutaneous, intrathecal, intraventricular,intramuscular, or intraperitoneal injection, or by intravenous (i.v.)drip); oral; topical (e.g., transdermal, sublingual, ophthalmic, orintranasal); or pulmonary (e.g., by inhalation or insufflation ofpowders or aerosols), or can occur by a combination of such methods.Administration can be rapid (e.g., by injection) or can occur over aperiod of time (e.g., by slow infusion or administration of slow releaseformulations).

Compositions and formulations for parenteral, intrathecal orintraventricular administration include sterile aqueous solutions (e.g.,sterile physiological saline), which also can contain buffers, diluentsand other suitable additives (e.g., penetration enhancers, carriercompounds and other pharmaceutically acceptable carriers).

Compositions and formulations for oral administration include, forexample, powders or granules, suspensions or solutions in water ornon-aqueous media, capsules, sachets, or tablets. Such compositions alsocan incorporate thickeners, flavoring agents, diluents, emulsifiers,dispersing aids, or binders.

Formulations for topical administration include, for example, sterileand non-sterile aqueous solutions, non-aqueous solutions in commonsolvents such as alcohols, or solutions in liquid or solid oil bases.Such solutions also can contain buffers, diluents and other suitableadditives. Pharmaceutical compositions and formulations for topicaladministration can include transdermal patches, ointments, lotions,creams, gels, drops, suppositories, sprays, liquids, and powders.Conventional pharmaceutical carriers, aqueous, powder or oily bases,thickeners and the like may be useful. Methods and compositions fortransdermal delivery include those described in the art (e.g., inWermeling et al. (2008) Proc. Natl. Acad. Sci. USA 105:2058-2063; Goebeland Neubert (2008) Skin Pharmacol. Physiol. 21:3-9; Banga (2007) Pharm.Res. 24:1357-1359; Malik et al. (2007) Curr. Drug Deliv. 4:141-151; andPrausnitz (2006) Nat. Biotechnol. 24:416-417).

Nasal preparations can be presented in a liquid form or as a dryproduct. Nebulized aqueous suspensions or solutions can include carriersor excipients to adjust pH and/or tonicity.

Pharmaceutical compositions include, but are not limited to, solutions,emulsions, aqueous suspensions, and liposome-containing formulations.These compositions can be generated from a variety of components thatinclude, for example, preformed liquids, self-emulsifying solids andself-emulsifying semisolids. Emulsion formulations are particularlyuseful for oral delivery of therapeutic compositions due to their easeof formulation and efficacy of solubilization, absorption, andbioavailability. Liposomes can be particularly useful due to theirspecificity and the duration of action they offer from the standpoint ofdrug delivery.

Compositions provided herein can contain any pharmaceutically acceptablesalts, esters, or salts of such esters, or any other compound which,upon administration to a subject, is capable of providing (directly orindirectly) the biologically active aptamer. The term “pharmaceuticallyacceptable salts” refers to physiologically and pharmaceuticallyacceptable salts of the nucleic acid aptamers useful in methods providedherein (i.e., salts that retain the desired biological activity of theparent aptamers without imparting undesired toxicological effects).Examples of pharmaceutically acceptable salts include, but are notlimited to, salts formed with cations (e.g., sodium, potassium, calcium,or polyamines such as spermine); acid addition salts formed withinorganic acids (e.g., hydrochloric acid, hydrobromic acid, sulfuricacid, phosphoric acid, or nitric acid); salts formed with organic acids(e.g., acetic acid, citric acid, oxalic acid, palmitic acid, or fumaricacid); and salts formed with elemental anions (e.g., bromine, iodine, orchlorine).

Compositions additionally can contain other adjunct componentsconventionally found in pharmaceutical compositions. Thus, thecompositions also can include compatible, pharmaceutically activematerials such as, for example, antipruritics, astringents, localanesthetics or anti-inflammatory agents, or additional materials usefulin physically formulating various dosage forms of the compositions, suchas dyes, flavoring agents, preservatives, antioxidants, opacifiers,thickening agents, and stabilizers. Furthermore, the composition can bemixed with auxiliary agents, e.g., lubricants, preservatives,stabilizers, wetting agents, emulsifiers, salts for influencing osmoticpressure, buffers, colorings, flavorings, penetration enhancers, andaromatic substances. When added, however, such materials should notunduly interfere with the biological activities of the other componentswithin the compositions.

In some cases, an aptamer provided herein can be formulated as asustained release dosage form. For example, an aptamer can be formulatedinto a controlled release formulation. In some cases, coatings,envelopes, or protective matrices can be formulated to contain one ormore of the polypeptides provided herein. Such coatings, envelopes, andprotective matrices can be used to coat indwelling devices such asstents, catheters, and peritoneal dialysis tubing. In some cases, anaptamer provided herein can be incorporated into a polymeric substances,liposomes, microemulsions, microparticles, nanoparticles, or waxes.

Pharmaceutical formulations as disclosed herein, which can be presentedconveniently in unit dosage form, can be prepared according toconventional techniques well known in the pharmaceutical industry. Suchtechniques include the step of bringing into association the activeingredient(s) (i.e., an aptamer) with the desired pharmaceuticalcarrier(s). Typically, the formulations can be prepared by uniformly andintimately bringing the active ingredient(s) into association withliquid carriers or finely divided solid carriers or both, and then, ifnecessary, shaping the product. Formulations can be sterilized ifdesired, provided that the method of sterilization does not interferewith the effectiveness of the molecules(s) contained in the formulation.

The nucleic acid aptamers (e.g., comprising SEQ ID NO:17) providedherein can be administered to a mammal (e.g., a human or a non-humanmammal) in order to reduce demyelination that can occur with diseasessuch as MS, for example. The aptamers can be administered at anysuitable dose, depending on various factors including, withoutlimitation, the agent chosen and the patient characteristics.Administration can be local or systemic.

In some embodiments, an aptamer or a composition containing an aptamercan be administered at a dose of at least about 0.01 ng/kg to about 100mg/kg of body mass (e.g., about 10 ng/kg to about 50 mg/kg, about 20ng/kg to about 10 mg/kg, about 0.1 ng/kg to about 20 ng/kg, about 3ng/kg to about 10 ng/kg, or about 50 ng/kg to about 100 μg/kg) of bodymass, although other dosages also may provide beneficial results.

The methods provided herein can include administering to a mammal aneffective amount of an aptamer, or an effective amount of a compositioncontaining such an aptamer. As used herein, the term “effective amount”is an amount of an aptamer or aptamer-containing composition that issufficient to reduce the occurrence of demyelination or increase theoccurrence of remyelination in a mammalian recipient by at least 10%(e.g., 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%,95%, 99%, or 100%). The presence or extent of demyelination andremyelination can be evaluated using methods known in the art,including, for example, the methods described in the Examples sectionherein.

In some embodiments, for example, an “effective amount” of an aptamer asprovided herein can be an amount that reduces demyelination in a treatedmammal by at least 10% as compared to the level of demyelination in themammal prior to administration of the aptamer, or as compared to thelevel of demyelination in a control, untreated mammal.

The invention will be further described in the following examples, whichdo not limit the scope of the invention described in the claims.

EXAMPLES Example 1 Materials and Methods

Preparation of Crude Murine Myelin:

CNS tissue from strain SJL mice (5 g) was homogenized in 0.32 M sucrosecontaining 2 mM EGTA (pH 7.5) using a tissue grinder, followed by aDounce homogenizer to yield a final volume of 100 ml. The homogenate (17ml) was layered onto 3 ml of 0.85 M sucrose containing 2 mM EGTA andsubjected to centrifugation at 28,000 rpm for 1 hour at 4° C. Materialwas collected from the interface and homogenized in a total volume of240 ml of a solution containing 2 mM EGTA. After centrifugation, thepellet was homogenized in 5 ml of a solution containing 10 mM EGTA, thevolume was brought to 400 ml in 10 mM EGTA, and the solution was stirredfor 15 minutes at 4° C. After centrifugation for 15 minutes at 10,000rpm, the homogenization and centrifugation steps were repeated. Theresulting pellet was homogenized in 100 ml of solution containing 0.85 Msucrose and 2 mM EGTA. The homogenate was overlayed with 3 ml of asolution containing 0.32 M sucrose and 2 mM EGTA, and subjected tocentrifugation at 28,000 rpm for 90 minutes. After repeatedhomogenization and washing, the myelin was isolated from the 0.32 M/0.75M interface of a discontinuous sucrose gradient, washed with distilledwater, and resuspended in 50 mM Tris-HCl containing 2 mM EGTA.

In Vitro Selection of DNA Aptamers:

A random DNA library was created and single-stranded 5′-fluorescein-(FIG. 2A) conjugated oligonucleotides were subjected to in vitroselection for binding to suspensions of crude murine myelin. The initialround of selection employed 2.5 nmole (˜1×10¹⁵ molecules) of randomoligonucleotide library LJM-2772. Oligonucleotides were heated to 90° C.for 1 minute in PBS containing 1 mM MgCl₂, placed on ice for 15 minutes,and then incubated for 8 minutes at room temperature to allow folding.200 μL of mouse myelin suspension (10 μg) was pelleted by centrifugationfor 5 minutes at 6500 rpm (microcentrifuge). The pellet was washed twiceby resuspension in 500 μL binding buffer (20 mM Tris-HCl, pH 7.6, 10 mMNaCl, 0.5 mM KCl). The DNA library (5 μM in the first round, 300 nM insubsequent rounds) was then incubated for 30 minutes with gentleagitation in a 500 μL binding reaction with 10 μg myelin suspension in500 μL binding buffer. The suspension and bound aptamers were washedtwice with 1 mL binding buffer by 6500 rpm centrifugation. To the pelletwas added 400 μL 2×PK buffer (300 mM NaCl, 2.5 mM EDTA, 2% SDS),followed by agitation, and extraction with 400 μL phenol:chloroform(1:1, v:v). DNA was precipitated from the aqueous phase by addition ofethanol. A portion of the recovered DNA was amplified by PCR toestablish the optimal number of amplification cycles. After the firstround, PCR was performed with a fluorescein-labeled primer, allowingquantitation of library recovery by fluorescence spectroscopy. The upperprimer sequence was 5′-F-ATACCAGCTTATTCAATT (SEQ ID NO:1; F indicatesfluorescein). The lower primer sequence was5′-AAAAAAAAAAAAAAAAAAAAXXAGATT GCACTTACTATCT (SEQ ID NO:2; X indicatesGLEN Research spacer phosphoramidite 10-1909). PCR reactions (100 μL)employed Taq DNA polymerase, primers at 10 μM final concentration, andincubation for 5 minutes at 94° C., followed by cycles of 30 seconds at94° C., 30 seconds at 47° C., and 30 seconds at 72° C. A second aliquotof recovered DNA was then amplified for the optimum number of cycles toprepare aptamer for the next selection round. Single-strandedfluorescent aptamer was obtained by precipitation of PCR reactions fromethanol, followed by denaturing polyacrylamide gel electrophoresis. Thefluorescent DNA band was cut from the gel, diced, and eluted in TEbuffer at 37° C. for 2-12 hours, followed by precipitation from ethanoland quantitation by UV spectrometry. After 11 selection cycles, PCR wasperformed and the resulting duplex DNA was ligated into the pGEM-Teasycloning vector (Promega, Madison, Wis.), cloned, and sequenced. Anactive 40-nucleotide sub-sequence (“3064,” having the sequence5′-GGGTCGGCGGGTGGGGTGGG AGGTGGTCTTGTCTCTGGGT-3′; SEQ ID NO:17); wasidentified for further study.

Aptamer Specificity Characterization:

To promote intramolecular folding, aptamer stock solutions (5 μM) in PBSwere heated to 90° C., MgCl₂ was added to a final concentration of 1 mM,and solutions were allowed to cool room temperature. Myelin stock wasdiluted in PBS and sonicated on ice. Fluorescent folded aptamers wereadded to different amounts of myelin suspension and incubated at 37° C.for 3 hours. Insoluble material with bound aptamers was recovered bycentrifugation for 30 seconds in a microcentrifuge. The pellet waswashed with 100 μL PBS and again recovered by centrifugation. Afterphenol extraction and ethanol precipitation, fluorescent aptamers werequantitated in black plastic 96-well plates using a Typhoon FluorescentImaging system (GE Healthcare Biosciences; Piscataway, N.J.). ForSouthwestern blotting, 15 μg crude myelin protein was separated in eachlane of a 10% Bis-Tris SDS polyacrylamide gel in2-(N-morpholino)ethanesulfonic acid (MES) buffer. After electrophoresis,duplicate lanes were either stained with Coomassie blue dye ortransferred to polyvinylidene fluoride (PVDF) membrane byelectroblotting. Western blotting was performed by standard methodsusing antibodies with the indicated specificities. For southwesternblots, membranes were blocked for 30 minutes at 37° C. in tris-bufferedsaline and TWEEN® 20 (TBST) buffer containing with 1% bovine serumalbumin (BSA), 10% non-fat dry milk, 2 mg/mL sonicated andheat-denatured salmon testis DNA, and TWEEN® 20 detergent. Foldedfluorescent aptamers (5 μM final concentration) were then added in PBScontaining 1 mM MgCl₂ and incubated with PVDF membranes for 14 hours,followed by washing in TBST buffer and then in 0.5× tris/borate/EDTA(TBE) buffer. Fluorescein fluorescence was then detected on membranesusing the Typhoon Fluorescent Imaging system. Tryptic peptide massfingerprinting was performed.

Mouse Model:

Eight-week-old female SJL/J mice (Jackson Laboratories; Bar Harbor, Me.)received a single intracerebral injection of 2×10⁵ plaque-forming unitsof the Daniel's strain of Theiler's Myeloencephilitus Virus (TMEV) inDulbecco's phosphate buffered saline (DPBS; 10 μL). The resultingencephalitic-like infection resulted in greater than 98% incidence ofdemyelination with increasing neurologic deficits progressing overseveral months (Rodriguez et al. (1987) Crit. Rev. Immunol. 7:325-365).Animals used for remyelination studies were chronically demyelinated bysix months post infection, with clear neurologic deficits. To assembletreatment groups in cages of five mice each, all mice to be treated werecombined in a large container and then distributed equally based on thelevel of disability. The extent of mouse disability was determined byexamination of the mouse coat color, a reflection of the ability toself-groom, gait, and the ability to right when placed on the dorsalside.

Aptamer Treatment:

DNA oligonucleotides 3064 (SEQ ID NO:17), 3060(5′-AAAGAACAAAAAGGATAAAGGGGGAGACGGGGGGAACATGGGG-3′; SEQ ID NO:20) and3202 (5′-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT-3′; SEQ ID NO:21) weresynthesized DMT-off at 1 μmol scale using 3′ biotinTEG control poreglass support (Glen Research 20-2955). Oligonucleotides were cleavedfrom the support and deprotected in hot ammonia, then dried and purifiedby reverse phase HPLC and sterilized by precipitation from ethanol.

Demyelinating CNS lesions were induced by infection using Daniel'sstrain of Theiler's virus in SJL mice (FIG. 7). After 27 weeks, micewere injected intraperitoneally (i.p.) with synthetic DNA aptamers (testsequences or controls) that had been prepared as 3′ biotinylated (FIG.2B) derivatives and mixed with excess streptavidin to promote stability.Groups of mice received i.p. 500-μL injections of the 3′ biotinconjugated aptamer (1 μM) combined with streptavidin (0.25 μM) incalcium-free D-PBS (Invitrogen; Carlsbad, Calif.) supplemented withmagnesium chloride (1 mM). Injections were given twice per week for fiveweeks. Briefly, sterile aptamer solution (1 μM) in calcium-free D-PBSsupplemented with MgCl₂ (1 mM) was heated to 90° C. for 1 minute, placedon ice for 15 minutes, and then incubated for 8 minutes at roomtemperature to allow aptamer folding. Streptavidin stock solution(Jackson Immune Research; West Grove, Pa.; 1 mg/mL; 18 μM incalcium-free D-PBS) was added to a final concentration of 0.25 μM andincubated with gentle agitation for 30 minutes at 37° C. immediatelyprior to i.p. injection into mice. The final aptamer injection solution(500 μL) contained streptavidin: 13.8 μg/mL (0.25 μM),aptamer-3′-Biotin: 13.4 (12.7-14.2) μg/mL (1.2-1.5 μM) in calcium-freeD-PBS supplemented with MgCl₂ (1 mM). Each treatment therefore consistedof 6.9 μg (125 pmol) streptavidin, 6.7 μg (500 pmol) aptamer-3′-biotin,and 47.6 μg (500 μmol) MgCl₂. Neuropathology to characterize the extentof lesion remyelination was performed five weeks after completion ofaptamer injections according to a blinded protocol.

Spinal Cord Morphometry:

Mice were anesthetized with sodium pentobarbital and perfusedintracardially with Trump's fixative (phosphate-buffered 4%formaldehyde/1% glutaraldehyde, pH 7.4). Spinal cords were removed andcut into 1 mm blocks, and every third block was postfixed and stainedwith osmium tetroxide and embedded in araldite plastic (Polysciences,Warrington, Pa.). One-micrometer-thick cross-sections were cut from eachblock, mounted onto glass slides, and stained with 4%praraphenylene-diamine to visualize myelin (Rodriguez et al. (1987) J.Immunol. 138: 3438-3442). Ten to twelve spinal cord sections,representing samples from the cervical, thoracic, lumbar, and sacralspinal cord, were stained with 4% p-phenylenediamine to visualize themyelin sheaths, and neuropathology was performed according to a blindedprotocol. Sections were analyzed for spinal cord pathology(inflammation, demyelination, remyelination) as previously described(McGavern et al. (1999) J. Neurosci. Res. 58:492-504). Each spinal cordsection was divided visually into four quadrants based on morphologicalsymmetry and examined by bright field microscopy at 100× and 200× totalmagnification using an Olympus Provis microscope. Each quadrant fromeach section was graded for the presence or absence of gray matterdisease, meningeal inflammation, and demyelination. Demyelinated areaswere characterized by denuded axons and inflammatory cell infiltrates.Demyelinated areas with remyelination were characterized by thin myelinsheaths compared with the thicker, intact myelin sheaths. The spinalcord white matter was scored as normal, demyelinated with noremyelination, or demyelinated with remyelination (Mitsunaga et al.(2002) FASEB J. 16:1325-1327). Partial quadrants were excluded. Lesionswere judged to be remyelinated when they were 75-100% repaired.Remyelinated lesions below this threshold were scored as negative. Datawere not assembled into treatment groups until all slides in a givenstudy were graded. Demyelination for each mouse was calculated as apercentage based on the number of spinal cord quadrants withdemyelination, which included quadrants with demyelination and repair,divided by the total number of quadrants scored. Remyelination for eachmouse was calculated as a percentage based on the number of demyelinatedquadrants above threshold remyelination divided by the number ofquadrants with demyelination. Data for percentage spinal corddemyelination and remyelination were compared across groups usingone-way ANOVA on ranks. When a significant difference (P<0.05) wasidentified, a pairwise comparison of aptamer-treated groups with theaptamer-control and untreated control groups was performed. Statisticalanalysis and plots were performed using SigmaStat and SigmaPlot. Dataare presented as mean±SEM.

Example 2 Identification and Characterization of Aptamers that BindMurine Myelin

In vitro selection from a single-stranded DNA library was used toidentify aptamers that bound to a suspension of crude murine myelin(FIG. 1A). The results of in vitro selection of DNA aptamer pools over10-11 rounds of selection and amplification are plotted in FIG. 3.Targets were myelin oligodendrocyte glycoprotein (MOG) immobilized onNi-NTA magnetic beads (filled circles) or suspension of crude mousemyelin in buffer (open circles). These procedures yielded a set of DNAmolecules that were sequenced (FIG. 4). MOG selections gave rise toaptamer 3060 (selective for chelated Nickel beads; FIG. 1D; SEQ IDNO:20). Myelin selections gave rise to aptamer 3064 (FIGS. 1B, 1C and 4;SEQ ID NO:17).

The anti-myelin aptamer 3064 was compared with negative control aptamers3060 and 3202 (SEQ ID NOS:20 and 21; FIGS. 1D and 1E). Interestingly, asobserved for some other DNA aptamers (Griffin et al. (1993) Gene137:25-31; and Somasunderam et al. (2005) Biochemistry 44:10388-10395;Andreola et al. (2001) Biochemistry 40:10087-10094), both aptamer 3064(specific for myelin) and control aptamer 3060 (selected for affinity tochelated nickel ions; Nastasijevic et al. (2008) Biochem. Biophys. Res.Commun. 366:420-425) contain guanosine-rich domains predicted to induceintra- or intermolecular folding through guanosine quartets.

The specificity of anti-myelin DNA aptamer 3064 and controls 3060 and3202 was analyzed by assessing the binding of fluorescent aptamers tocrude myelin protein suspension using centrifugal sedimentation torecover bound aptamers (FIG. 5A). Myelin-specific aptamer 3064 showedstrong myelin-dependent binding, while aptamers 3060 and 3202 did not.Because the myelin preparation was a crude mixture of proteins andlipids, however, these results did not provide quantitative affinityestimates. To further assess specificity, crude myelin proteins wereseparated by SDS-polyacrylamide gel electrophoresis and stained withCoomassie dye (myelin; FIG. 5B, left lane), or blotted to polyvinylidene(PVDF) membrane and probed with antibodies against myelin basic protein(MBP), proteolipid protein (PLP), or MOG in western blots (FIG. 5B,lanes 2-4), or probed with fluorescent aptamers 3202, 3060 or 3064 insouthwestern blots (FIG. 5B, lanes 5-7). While control aptamer 3202showed no binding to myelin proteins, guanosine-rich aptamers 3060 and3064 bound specifically to certain proteins (FIG. 5B). In particular,control aptamer 3060 and myelin-specific aptamer 3064 both bound tothree myelin proteins with apparent molecular weights of 12.5 kDa, 18.5kDa, and 23.8 kDa. Based on western analysis and mass spectrometry oftryptic peptides, these proteins were PLP, the 18.5 kDa isoform of MBP,and MOG, respectively. Myelin-specific aptamer 3064 uniquely bound tomyelin proteins with apparent molecular weights of 17 kDa and 21.5 kDa,identified as MBP isoforms containing sequences encoded by MBP exon 2(Campagnoni (1988) J. Neurochem. 51: 1-14).

DNA aptamer 3064 was observed to bind preferentially to an intact myelinsuspension (FIG. 5A), while both aptamers 3060 and 3064 showed affinityfor certain myelin proteins after extraction of lipids andimmobilization (FIG. 5B). These results suggested that MBP sequencesencoded in exon 2 were targeted by aptamer 3064, and these sequenceswere preferentially accessible in crude myelin suspensions. Extractionof proteins for Southwestern blotting revealed other aptamer bindingsites that may not be accessible in intact myelin.

The specificity of anti-myelin DNA aptamer 3064 and controls 3060 and3202 also was analyzed by assessing binding of fluorescent aptamerderivatives to living murine cerebellar sections (FIG. 6). Negativecontrol aptamer 3202 showed little or no binding to cerebellar sections,control aptamer 3060 bound diffusely to white matter, and anti-myelinaptamer 3064, which bound to crude myelin in vitro, strongly boundmyelin-rich regions of the brain (FIG. 6).

Example 3 In Vivo Enhancement of Remyelination by DNA Aptamer Treatment

The binding properties of anti-myelin DNA aptamer 3064 were similar tocertain natural human IgM autoantibodies that promote remyelination inthe murine Theiler's encephalomyelitis virus model of MS (Warrington etal., supra). Intraperitoneal injection of anti-myelin DNA aptamer 3064therefore was compared to negative control aptamers 3060 and 3202.Aptamers were prepared as 3′-biotin conjugates (FIG. 2B) and incubatedin a 4:1 molar ratio with tetrameric streptavidin to create conjugateswith enhanced stability and biodistribution (Dougan et al. (2000)Nuclear Med. Biol. 27:289-297), mimicking polyvalent antibodies. Asshown in FIG. 8 (lanes 2, 6, and 10), 4:1 aptamer:streptavidinincubation under these conditions converted all monomeric aptamers tostreptavidin complexes, predominantly dimers.

Aptamers were injected into mice 27 weeks after Theiler's virusinfection, and CNS pathology was assessed by blinded review ofneuropathology after 5 weeks. Results are shown in Table 1 and FIG. 9A.Anti-myelin DNA aptamer 3064 induced remyelination (with at least 75% ofthe lesion being remyelinated) in 26% of experimental CNS lesions,compared to 8% and 4% for negative control aptamers 3060 and 3202,respectively. These statistically significant results indicatedselective enhancement of remyelination in vivo by anti-myelin DNAaptamer treatment.

TABLE 1 Remyelination in vivo after DNA aptamer treatment AptamerQuadrants with Demyelinated quadrants (specificity) n¹ demyelination (%)with remyelination (%) 3060 (anti-Ni) 8 45.8 ± 6.9 8.3 ± 3.9 3202(oligo-dT) 8 56.4 ± 6.3 3.7 ± 2.1 3064 (anti-myelin) 6 41.8 ± 7.5 26.2 ±7.3  ¹Number of mice in each treatment group

Statistical Comparisons:

3064 to 3202, p=0.008

3064 to 3060, p=0.043

3060 to 3202, p=0.505

All statistics by comparing groups using rank order sum test.

Further experiments were conducted to compare the effect of aptamersadministered to mice in the presence or absence of biotin and avidin. Asabove, the Theiler's virus model of MS was used for these experiments.Mice were treated 6 to 9 months after infection—a time point of maximaldemyelination and minimal remyelination. All data were collected oncoded samples where the only information was the animal number on theslide. Spinal cords from all mice in the study contained areas ofchronic demyelination. Infiltrating macrophages were present in severallesions. Remyelination was characterized by densely packed thin myelinsheaths in relation to axon diameter. In mice treated with anti-myelinaptamer 3064, more areas of dense remyelinated axons were found (FIG.9B). For example, the top middle panel of FIG. 9B shows an area ofalmost complete remyelination mediated by oligodendrocytes located inthe dorsal white matter column of the spinal cord of a mouse treatedwith anti-myelin aptamer 3064. In mice treated with control aptamers3060 and 3202, dorsal column spinal cords lesions contained fewerremyelinated axons.

As indicated in Table 2 below, mice that received aptamer 3064(anti-myelin) with the presumed tetrameric structure showedremyelination in 35% of experimental CNS lesions, compared to 4% and 9%remyelination after treatment with negative control aptamers 3202 (dT₄₀)and 3060 (anti-Ni), respectively. Animals that received aptamer 3064alone (without biotin-avidin) showed remyelination similar to thecontrols. These statistically significant results suggested that theaptamer must be in the “tetrameric” structure in order to promoteremyelination in vivo. Interestingly, animals injected with streptavidinand non-biotinylated aptamers 3064 or 3060 did not show remyelination,suggesting that aptamer 3′ modification was important for protectionfrom nuclease attack and/or for streptavidin binding to form multivalentconjugates.

TABLE 2 Remyelination in vivo after treatment with DNA aptamers with andwithout biotin-avidin Gray Inflam- Demyelin- Remyelin- Treatment N*Matter mation ation ation 3064 w/ 10 0.0 ± 0.0  6.0 ± 3.1 41.1 ± 6.434.9 ± 6.1  biotin-avidin 3060 w/ 7 0.0 ± 0.0 14.5 ± 6.2 38.2 ± 8.7 8.8± 4.5 biotin-avidin 3202 w/ 8 0.0 ± 0.0  3.5 ± 1.1  51.8 ± 26.4 4.2 ±2.3 biotin-avidin 3064 w/o 9 0.0 ± 0.0 13.1 ± 6.6 42.0 ± 5.8 8.5 ± 3.4biotin-avidin 3060 w/o 8 0.0 ± 0.0 30.5 ± 7.3   50 ± 4.6 10.3 ± 4.4 biotin-avidin *N = number of miceTable indicates lesion status, by animal, after blinded review ofneuropathology. Data are expressed as the percent of spinal cordquadrants showing that at least 75% of the lesion was remyelinated(mean±SEM).

Statistical Comparisons:

p<0.002 for remyelination—(ANOVA on Ranks); Dunn's comparison to dT₄₀(control) showed statistical difference (p<0.05) against themyelin-reactive 3064 aptamer with biotin/avidin. There was nosignificant difference in remyelination between antisense (control) andthe myelin reactive 3064 aptamer without biotin-avidin.

Further experiments with additional mice confirmed these results. Datafrom all of the experiments are presented in Table 3A, with statisticalresults in Table 3B.

TABLE 3A Gray Inflam- Demyelin- Remyelin- Treatment N* Matter mationation ation 3064 15 0.0 ± 0.0 10.3 ± 3.0 39.4 ± 4.4 32.3 ± 4.3  wbiotin-avidin 3060 w/ 7 0.0 ± 0.0 14.5 ± 6.2 38.2 ± 8.7 8.8 ± 4.5biotin-avidin 3202 w/ 7 0.0 ± 0.0  3.5 ± 1.1  51.8 ± 26.4 4.2 ± 2.3biotin-avidin 3064 w/o 9 0.0 ± 0.0 13.1 ± 6.6 42.0 ± 5.8 8.5 ± 3.4biotin-avidin 3060 w/o 6 0.0 ± 0.0 30.5 ± 7.3   50 ± 4.6 10.3 ± 4.4 biotin-avidin Streptavidin 15 0.0 ± 0.0 17.2 ± 3.1 47.4 ± 3.4 10.7 ±2.7  only (no aptamer) No aptamer 6 0.0 ± 0.0 20.2 ± 4.5 46.2 ± 7.7 7.2± 2.5 *N = number of miceData are expressed as the percent of spinal cord quadrants showingpathologic abnormality (mean±SEM).

Statistical Comparisons:

p<0.001 remyelination—(ANOVA on Ranks); Dunn's comparison to dT₄₀(control) showed statistical difference (p<0.05) against themyelin-reactive 3064 aptamer with biotin/streptavidin. No significantdifference in remyelination was observed between dT₄₀ (control) andaptamer 3064 without biotin-streptavidin.

TABLE 3B Parameter Group Group Statistics Value Remyelination All ranksANOVAs p < 0.001 Demyelination All ranks ANOVAs p = 0.391 InflammationAll ranks ANOVAs p = 0.448 Remyelination 3064 w/biotin 3202 w/biotin Ttest p = 0.038 Demyelination 3064 w/biotin 3202 w/biotin T test p =0.156 Remyelination 3064 w/o biotin 3064 w/o biotin T test p = 0.002Remyelination 3064 w/o biotin Streptavidin T test p < 0.001 only

The aptamer-induced remyelination described herein was comparable toeffects obtained in studies using much larger and more labile human IgMautoantibodies. The present results raised the possibility that theobserved remyelination activity of anti-myelin DNA aptamers also mayreflect direct interactions with lesions, though this remains to bedemonstrated. The abilities of certain natural IgM autoantibodies tostimulate cell signaling and remyelination depend on the multivalentcharacter of the antibody structure (Paz Soldan, supra). It isnoteworthy that the formulation of biotinylated DNA aptamers withstreptavidin is likely to organize tetravalent complexes by virtue ofintermolecular guanosine quartets (see, e.g., FIG. 1C).

The molecular mass of anti-myelin DNA aptamer 3064 is ˜13,000. Evenfully tetramerized with streptavidin (mass ˜52,800), the resultingcomplex (mass ˜104,800) is still about 10-fold smaller than the IgMantibodies previously shown to promote remyelination. In addition, DNAaptamers can be prepared by chemical synthesis, and are less likely thanIgM antibodies to be immunogenic. These considerations suggest that DNAaptamer reagents may be useful as a therapeutic for treatment of MS andother demyelinating diseases.

Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

What is claimed is:
 1. A method for promoting neuronal remyelination ina subject in need thereof, comprising administering to the subject apharmaceutical composition comprising a multimeric nucleic acid aptamerin an amount effective to promote remyelination, wherein the nucleicacid aptamer comprises the sequence set forth in SEQ ID NO:17.
 2. Themethod of claim 1, wherein the nucleic acid aptamer is a homotetramer.3. The method of claim 1, wherein the remyelination is mediated bycentral nervous system-type myelin producing cells or by peripheralnervous system-type myelin producing cells.
 4. The method of claim 1,wherein the subject is diagnosed with a demyelinating disease.
 5. Themethod of claim 4, wherein the demyelinating disease is selected fromthe group consisting of multiple sclerosis, idiopathic inflammatorydemyelinating diseases, transverse myelitis, Devic's disease progressivemultifocal leukoencephalopathy, optic neuritis, leukodystrophies andacute disseminated encephalomyelitis, Guillain-Barré syndrome, chronicinflammatory demyelinating polyneuropathy, anti-MAG peripheralneuropathy, and Charcot-Marie-Tooth disease.
 6. A composition comprisinga pharmaceutically acceptable carrier and a nucleic acid aptamercomprising the sequence set forth in SEQ ID NO:17.
 7. The composition ofclaim 6, wherein the nucleic acid aptamer is a homotetramer.
 8. A methodfor treating a subject diagnosed as having a demyelinating disease,comprising administering to the subject a pharmaceutical compositioncomprising a multimeric nucleic acid aptamer, wherein the nucleic acidaptamer comprises the sequence set forth in SEQ ID NO:17.
 9. The methodof claim 8, wherein the multimeric nucleic acid aptamer is ahomotetramer.
 10. The method of claim 8, wherein the demyelinatingdisease is selected from the group consisting of multiple sclerosis,idiopathic inflammatory demyelinating diseases, transverse myelitis,Devic's disease progressive multifocal leukoencephalopathy, opticneuritis, leukodystrophies and acute disseminated encephalomyelitis,Guillain-Barré syndrome, chronic inflammatory demyelinatingpolyneuropathy, anti-MAG peripheral neuropathy, and Charcot-Marie-Toothdisease.